First report of Leishmania RNA virus 1 in Leishmania (Viannia) braziliensis clinical isolates from Rio de Janeiro State - Brazil

BACKGROUND Leishmania parasites carry a double-stranded RNA virus (Leishmania RNA virus - LRV) that has been divided in LRV1 and LRV2. OBJECTIVES Leishmania (Viannia) braziliensis clinical isolates were assessed in order to determine LRV presence. METHODS Two-round polymerase chain reaction (PCR and nested PCR) was performed to detect LRV1 or LRV2 in L. (V.) braziliensis clinical isolates (n = 12). FINDINGS LRV1 was detected in three clinical isolates which was phylogenetically related to other sequences reported from other American tegumentary leishmaniasis (ATL) endemic areas of Brazil. Patients infected with L. (V.) braziliensis LRV-negative showed only cutaneous lesions while LRV-positive reported different manifestations. MAIN CONCLUSION Data presented here show for the first time that LRV1 is circulating in L. (V.) braziliensis clinical isolates from Rio de Janeiro State in Brazil.

since it was demonstrated, on in vivo models, that coinoculation of L. (L.) mexicana and L. (V.) panamensis with their respective exosomes increased lesion size but co-inoculation with L. (V.) guyanensis LRV positive exosomes exacerbated lesion development. (28) Another study revealed that LRV1 positivity frequency in L. (V.) guyanensis and L. (V.) braziliensis isolates from patients with MCL was higher than in patients with CL. (23) However, the data on the subject is contradictory, as another study showed a similar LRV1 detection rate among L. (V.) braziliensis, L. (V.) guyanensis and L. (V.) peruviana metastatic and non-metastatic strains in a longitudinal cohort of ATL patients from Peru. (29) Also, in a cohort of ATL patients from the southeast, north and northeast regions of Brazil, less than 5% of strains were LRV1 positive and the severity of the disease was related to other factors such as age, gender and immune status of the hosts. (22) In another study with 40 L. (V.) braziliensis isolates from Minas Gerais State, no sample was positive for LRV1. (30) In this context, we report for the first time that L. (V.) braziliensis clinical isolates from ATL patients living in Rio de Janeiro State can be infected with LRV1. In fact, the results presented here contribute to reinforce the heterogeneity previously seen for these clinical isolates. (31,32)

MATERIALS AND METHODS
In this study, RNA was obtained from stationaryphase promastigotes (10 7  ) guyanensis], cultured in vitro as previously described. (31) Each sample was lysed in TRIzol containing chloroform, and RNA was extracted using RNeasy Mini Kit (QIA-GEN, Germany). Then, RNA samples were converted into cDNA, using High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, USA), to detect LRV1 and LRV2 with specific primers in a two-round polymerase chain reaction (PCR) ( Table I). Both PCR and nested PCR were conducted in a final volume of 25 µL of reaction containing 1X PCR buffer, 3 mM of MgCl 2 , 2.5 U of Taq DNA Polymerase (Invitrogen Life Technologies, Brazil), 200 mM of triphosphate deoxyribonucleotides dNTP (Invitrogen Life Technologies, Brazil) and 0.2 µM of each primer, and the PCR assay conditions were performed as described in Table I. The amplicons obtained from nested PCR were purified using the NucleoSpin ® Gel and a PCR Clean-up kit (Macherey-Nagel GmbH & Co. KG, Germany), with a minor change in incubation (increased to five minutes). The purified products were subjected to sequencing in both directions in triplicate using the ABI Prism™ BigDye Terminator Cycle Sequencing kit (Applied Biosystems, USA) on an ABI 3730 automatic DNA sequencer at Fiocruz facilities [Capillary Electrophoresis DNA Sequencing Platform (SANGER) -RPT01A]. (33) The obtained sequences were deposited in GenBank under accession number ON409677-ON409679. Electropherograms were analysed using Chromas 2.4, while percent identity with sequences producing significant alignments was performed using the Basic Local Alignment Search Tool using nucleotide (BLASTn). Nucleotide sequences were aligned by the CLUSTAL W algorithm from Mo-  lecular Evolutionary Genetics Analysis (MEGA) X. (34) The phylogenetic analysis was performed using MEGA and the range estimation equations used were JIN and NEI (Kimura 2-parameter model). For the LRV positive samples, previously characterised Leishmania Viannia species were also confirmed by SANGER sequencing using HSP70 gene. (35)

RESULTS
The presence of LRV1 was identified in three L. (V.) braziliensis isolates from patients living in Rio de Janeiro by gel electrophoresis (Fig. 1) and confirmed by sequencing. Phylogenetic analysis indicates that the LRV1 have greater identity compared to other LRV1 identified in L. (V.) braziliensis isolated from patients of other Brazilian cities, such as Porto Velho and Candeias of Rondonia State (Fig. 2).

DISCUSSION
In accordance with LRV reported by other authors in Latin America, LRV2 was not identified in this study. Although all the individuals were residents of Rio de Janeiro, one of the patients was infected by L. (V.) braziliensis presumably in the municipality of Carangola in Minas Gerais State (Table II), where the presence of LRV has been previously reported in patients with CL. (36) The infection by L. (V.) braziliensis of the other two patients in the study occurred in the state of Rio de Janeiro (municipalities of Barra Mansa and Rio de Janeiro) (Table II), where circulation of LRV has not been reported so far. In addition, to confirming LRV1, it was possible to observe the following single-nucleotide polymorphisms among the isolates: T/A (position 35, isolates 7 and 11 versus 12), T/G (position 37, isolates 7 and 11 versus 12), T/C (position 65 isolates 7 and 12 versus 11), G/A (position 71, isolates 7 and 12 versus 11). Moreover, it is important to mention that these three clinical cases have a more exuberant disease profile, and at least two needed subsequent treatments, while all the negative cases are associated to patients with CL (Table II).
In conclusion, the findings presented here are original and serve as an alert that LRV1 is circulating in L. (V.) braziliensis in Rio de Janeiro. Additional studies with more clinical isolates are needed to assess a possible correlation between LRV1-infected parasites, clinical manifestations and treatment response, as described for ATL in other endemic areas.